RNA-Mediated Gene Modulation

ABSTRACT

An isolated RNA comprising an intron RNA that is released in a cell, thereby modulating the function of a target gene. Also disclosed are a composition comprising a chemokine and an isolated RNA of the invention or a DNA template for the isolated RNA, a composition comprising one or more agents that induce RNA-mediated modulation of the functions of two or more target genes in a cell, and methods of using these compositions for modulating the functions of genes in a cell.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/411,062, filed Sep. 16, 2002, and U.S. Provisional ApplicationSer. No. 60/418,405, filed Oct. 12, 2002, the contents of which areincorporated herein by reference.

FUNDING

This invention was made with support in part by a grant from NIH (CA85722). Therefore, the U.S. government has certain rights.

TECHNICAL FIELD

This invention relates to regulation of a gene function.

BACKGROUND

One strategy for treating human diseases is to target specificdisease-associated genes by either replacing impaired gene functions orby suppressing unwanted gene functions. Expression vectors are commonlyused for introducing active genes into a cell to provide missing genefunctions. To suppress unwanted gene functions, antisenseoligonucleotides, antibodies, and small molecule drugs are often used astherapeutic agents.

Applications of RNA interference (RNAi) (Elbashir et al. (2001) Nature411: 494-498) and deoxyribonucleotidylated-RNA interfering (D-RNAi) (Linet al. (2001) Biochem. Biophys. Res. Commun. 281: 639-644) technologiesin treating human diseases are also in progress. RNAi elicitspost-transcriptional gene silencing (PTGS) phenomena, knocking downspecific gene expression with high potency and less toxicity thantraditional antisense gene therapies. However, the gene silencingeffects mediated by dsRNA are repressed by interferon-induced global RNAdegradation when the dsRNA size is larger than 25 base pairs (bp),especially in mammalian cells. Although transfection of shortinterfering RNA (siRNA) or microRNA (miRNA) of less than 21 bp canovercome interferon-associated problems, the size limitation impairs theusefulness of RNAi, as it is difficult to deliver such small andunstable dsRNAs in vivo due to high dsRNase activities in human bodies(Brantl (2002) Biochimica et Biophysica Acta 1575: 15-25). Therefore,there remains a need for a more effective and reliable gene modulationsystem.

SUMMARY

This invention is based, at least in part, on the discovery that anartificial intron can be used to regulate the function of a gene in acell.

In one aspect, the invention features an isolated RNA comprising anintron RNA. The intron RNA is released in a cell (e.g., a mammaliancell), thereby modulating the function of a target gene. The isolatedRNA does not contain a combination of a splice donor site that includes5′-GU(A/G)AGU-3′ and a splice acceptor site that includes5′-CU(A/G)A(C/U)NG-3′ (N is A, G, C, or U). It may contain a splicedonor site that includes 5′-GUA(A/-)GAG(G/U)-3′ (“-” designates an emptyposition), a splice acceptor site that includes5′-G(A/U/-)(U/G)(C/G)C(U/C)(G/A)CAG-3′ (SEQ ID NO:1), a branch site thatincludes 5′-UACU(A/U)A(C/U)(-/C)-3′, a poly-pyrimidine tract thatincludes 5′-(U(C/U))₁₋₃(C/-)U₇₋₁₂C(C/-)-3′ (SEQ ID NO:2) or5′-(UC)₇₋₁₂NCUAG(G/-)-3′ (SEQ ID NO:3), or a combination thereof. Forexample, the splice donor site can be 5′-AGGUAAGAGGAU-3′ (SEQ ID NO:4),5′-AGGUAAGAGU-3′ (SEQ ID NO:5), 5′-AGGUAGAGU-3′, or 5′-AGGUAAGU-3′; thesplice acceptor site can be 5′-GAUAUCCUGCAGG-3′ (SEQ ID NO:6),5′-GGCUGCAGG-3′, or 5′-CCACAGC-3′; and the branch site can be5′-UACUAAC-3′ or 5′-UACUUAUC-3′. The isolated RNA can be introduced intoa cell for control of a gene function.

The invention also provides a DNA template for the isolated RNA of theinvention, an expression vector comprising the DNA, a cultivated cellcomprising the isolated RNA or the DNA, an animal (e.g., a mammal suchas a mouse) comprising the isolated RNA or the DNA, and a compositioncomprising the isolated RNA or the DNA.

The invention further provides a method of producing an intron RNA. Themethod comprises cultivating the above-described cell to allowexpression and/or release of the intron RNA. The released intron RNA canbe left in the cell for control of a gene function, or be collected fromthe cell and used for generation of a DNA-RNA hybrid or delivery intoanother cell.

Also within the scope of the invention is a method of modulating thefunction of a target gene in a cell. The method comprises introducinginto a cell an effective amount of the isolated RNA or DNA of theinvention. The intron RNA is then released in the cell, therebymodulating the function of a target gene.

In another aspect, the invention features a composition comprising achemokine (e.g., interleukin-2) and an isolated RNA or a DNA asdescribed above. An effective amount of this composition can beadministering into a cell (e.g., a mammalian cell or a cell infected bya virus) to modulate the function of a target gene. For example, anHIV-1-infected cell can be treated with a combination of interleukin-2and an isolated RNA containing an intron RNA complementary to an HIV-1genomic sequence. The intron RNA induces degradation of the HIV-1genomic sequence or its derivatives, or prevent it from being translatedinto polypeptides.

In still another aspect, the invention features a composition comprisingone or more agents that induce RNA-mediated modulation of the functionsof two or more target genes in a cell (e.g., a mammalian cell or a cellinfected by a virus). A method of modulating the functions of genes in acell by administering into the cell an effective amount of thecomposition is also within the scope of the invention. For example, whena cell is infected by HIV-1, it can be treated with one or more DNA-RNAhybrids or exogenous intron RNAs that cause degradation of HIV-1 RNAs,cellular RNAs such as Naf1β, Nb2HP, and Tax1BP RNAs, or theirderivatives, or prevent these RNAs from being translated intopolypeptides.

The present invention provides compositions and methods for treatinghuman diseases. Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. In case ofconflict, the present document, including definitions, will control.Preferred methods and materials are described below, although methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. Thematerials, methods, and examples disclosed herein are illustrative onlyand not intended to be limiting. Other features, objects, and advantagesof the invention will be apparent from the description and theaccompanying drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a novel strategy for producing desired RNA molecules incells through RNA splicing.

FIG. 2 depicts generation of antisense RNAs by spliceosome cleavage ofretroviral (e.g., LTR) promoter-mediated precursor transcripts.

FIG. 3 depicts generation of sense and antisense siRNAs by spliceosomecleavage of viral (e.g., CMV or AMV) promoter-mediated precursortranscripts.

FIG. 4 depicts generation of hairpin RNAs by spliceosome cleavage of PolII (e.g., TRE or Tet response element) promoter-mediated precursortranscripts.

FIG. 5 depicts microscopic results, showing interference with greenfluorescent protein (eGFP) expression in rat neuronal stem cells byvarious SpRNAi constructs.

FIG. 6 depicts Western blot results, showing interference with greenfluorescent protein (eGFP) expression in rat neuronal stem cells byvarious SpRNAi constructs.

FIG. 7 depicts Western blot results, showing interference with integrinβ1 (ITGb1) expression in human prostatic cancer LNCaP cells by variousSpRNAi constructs.

FIGS. 8A-B depict Northern blot analysis of SpRNAi-induced cellular genesilencing against HIV-1 infection (n=3).

FIG. 9 depicts potential differences between traditional PTGS/RNAi andSpRNAi phenomena.

FIGS. 10A-B depict experimental evidence for generation ofD-RNAi-induced miRNA.

FIGS. 11A-C depict in vivo gene silencing by anti-β-catenin D-RNAi inembryonic chicken.

FIG. 12 depicts in vivo gene silencing by anti-tyr D-RNAi in mouse.

DETAILED DESCRIPTION

This invention relates to RNA-mediated gene modulation. FIG. 1 shows anexample of a novel strategy for producing a desired RNA molecule in acell after RNA splicing event occurs. The desired RNA molecule, like anatural intron, is flanked by an RNA splicing donor and an acceptorsite. The DNA template for the desired RNA is inserted into a gene,which is expressed by type-II RNA polymerase (Pol II) transcriptionmachinery under the control of either Pol II or viral RNA promoter. Uponintracellular transcription, the transcript so produced is subjected toRNA splicing and/or processing events, thereby releasing the desired RNAmolecule. In certain cases, the desired RNA molecule is an antisense RNAthat serves as an antisense oligonucleotide probe for antisense genetherapy. In other cases, the desired RNA molecule is translated into apolypeptide that is useful in gene replacement therapy. The desired RNAmolecule can also consist of small antisense and sense RNA fragments tofunction as double-stranded siRNA for RNAi induction. Moreover, thedesired RNA molecule can be a small hairpin-like RNA capable of causingRNAi-associated gene silencing phenomena. In addition, the desired RNAmolecule can also be a ribozyme. All of the above desired RNA moleculesare produced by intracellular splicing events and therefore named as“SpRNAi” for convenience.

Accordingly, the invention features an isolated RNA comprising an intronRNA that is released in a cell, thereby modulating the function of atarget gene. An “isolated RNA” is a ribonucleic acid the structure ofwhich is not identical to that of any naturally occurring ribonucleicacid or to that of any fragment of a naturally occurring ribonucleicacid. A “function of a target gene” refers to the capability of thetarget gene to be transcribed into an RNA, the capability of the RNA tobe stabilized, processed (e.g., through splicing), reverse transcribedor translated, and the capability of the RNA to play its normal role,e.g., serving as a tRNA and rRNA.

RNA splicing is a process that removes introns and joins exons in aprimary transcript. The structures of intron RNAs are well known in theart. An intron usually contains signal sequences for splicing. Forexample, most introns start from the sequence GU and end with thesequence AG (in the 5′ to 3′ direction), which are referred to as thesplice donor and splice acceptor site, respectively. In addition, anintron has a branch site between the donor and the acceptor site. Thebranch site contains an A residue (branch point), which is conserved inall genes. In many cases, the exon sequence is (A/C)AG at the5′-exon-intron junction, and is G at the 3′-exon-intron junction. Thefourth element is a poly-pyrimidine tract located between the branchsite and the acceptor site.

In an isolated RNA of the invention, the splice donor site may contain5′-GUA(A/-)GAG(G/U)-3′, the splice acceptor site may contain5′-G(A/U/-)(U/G)(C/G)C(U/C)(G/A)CAG-3′, a branch site may contain5′-UACU(A/U)A(C/U)(-/C)-3′, and a poly-pyrimidine tract may contain5′-(U(C/U))₁₋₃(C/-)U₇₋₁₂C(C/-)-3′ or 5′-(UC)₇₋₁₂NCUAG(G/-)-3′.Functionally equivalents of these sequences (e.g., sequences containingmodified nucleotides) are included in the invention. The intron RNAserves as or is farther processed to become, e.g., an RNA encoding apolypeptide, or an antisense RNA, short-temporary RNA (stRNA), microRNA(miRNA), small-interfering RNA (siRNA), short-hairpin RNA (shRNA), longdeoxyribonucleotide-containing RNA (D-RNA), or ribozyme RNA, each ofwhich may be in either sense or antisense orientation. Design ofantisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA and ribozyme RNA iswell known in the art. The intron RNA region homologous or complementaryto its target gene ranges from 14 to 2,000 nucleotides, most preferablybetween 19 and 500 nucleotides. The intron RNA may be 35-100% (i.e., anyintegral between and including 35 and 100) identical or complementary toits target gene. The preferred homology or complementarity is 35-65% andmore preferably 41-49% for an shRNA, 40-100% and more preferably 90-100%for a sense or antisense RNA. The length of an siRNA/miRNA/shRNA may be16-38 nucleotides, and preferably 19-25 nucleotides. Additionally, theremay be one or more linker sequences, e.g., between the donor and theacceptor site and the antisense RNA, stRNA, siRNA, shRNA, D-RNA orribozyme RNA sequence. The isolated RNA may further contain exonsencoding a polypeptide for co-expression with the intron RNA. Thepolypeptide may be a normal protein, a missing protein, adominant-negative protein, or a protein marker such as a fluorescentprotein, luciferase, or lac-Z.

An isolated RNA of the invention can be chemically synthesized orproduced by transcription from a DNA template in vitro and in vivo. Thetemplate DNA can be cloned into an expression vector according themethods well known in the art. Examples of such vectors include, but arenot limited to, plasmids, cosmids, phagemids, yeast artificialchromosome, retroviral vectors, lentiviral vectors, lambda vector,adenoviral (AMV) vector, adeno-associated viral (AAV) vector, hepatitisvirus (HBV)-modified vector, and cytomegalovirus (CMV)-related viralvector.

The isolated RNA, DNA template, and expression vector described abovecan be introduced into a cultured cell or a subject (e.g., an animal ora human) using methods commonly employed in the art such astransfection, infection, electroporation, micro-injection, and gene-gunpenetration. To help with the delivery into a cell, the isolated RNA,DNA template, and expression vector may be formulated into acomposition. The intron RNA, once expressed and/or released in the cell,can modulate the function of a target gene, for example, inhibit acancer-related gene, potential viral gene, and microbe-related gene.Therefore, this method is useful for treating and preventing diseasessuch as cancer and viral or microbial infection.

In one in vivo approach, a composition is suspended in apharmaceutically acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous infusion, or injected or implantedsubcutaneously, intramuscularly, intrathecally, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thesubject's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100.0 μg/kg. Widevariations in the needed dosage are to be expected in view of thevariety of compounds available and the different efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by i.v.injection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the compound in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Alternatively, the composition may be delivered to the subject, forexample, by use of polymeric, biodegradable microparticle ormicrocapsule delivery devices known in the art.

Another way to achieve uptake of the nucleic acid is to use liposomes,prepared according to standard methods. The vectors can be incorporatedalone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano etal. (1995) J. Mol. Med. 73: 479). Furthermore, tissue specific targetingcan be achieved by use of tissue-specific transcriptional regulatoryelements (TRE) which are known in the art. Delivery of naked nucleicacids (i.e., without a delivery vehicle) to an intramuscular,intradermal, or subcutaneous site is another means to achieve in vivoexpression.

An “effective amount” is an amount of the compound or composition thatis capable of producing a medically desirable result (e.g., a decreasedexpression level of a cancer-related gene, potential viral gene, ormicrobe-related gene) in a treated subject.

In particular, an animal comprising an isolated RNA or a DNA of theinvention can be produced according to the methods described above orany other methods known in the art. For example, a “knock-out animal”may be generated in which a target gene is partially (e.g., only in sometissues) or completely inhibited. The animal can be a farm animal suchas a pig, goat, sheep, cow, horse and rabbit, a rodent such as a rat,guinea pig, and mouse, or a non-human primate such as a baboon, monkey,and chimpanzee.

These animals of the invention can be used as disease models. Inparticular, these animals can be used to identify a compound orcomposition effective for treatment or prevention of a disease.Compounds or compositions can be identified by administering a testcompound or composition to a model animal or by contacting the testcompound or composition with an organ, a tissue or cells derived from amodel animal. Effects of the test compound or composition on the diseaseof the animal, organ, tissue or cells are evaluated. Test compounds orcompositions that palliate the disease symptoms can be effective fortreatment or prevention of the disease.

A second aspect of the invention is based on the discovery that thecombination of interleukin-2 and a viral RNA-antisense DNA hybridsignificantly reduced human immunodeficiency virus-1 (HIV-1) subtype Bgene activity. Consequently, the invention features a compositioncomprising a chemokine and an isolated RNA or a DNA of the invention.The isolated RNA or DNA allows an intron RNA to be released in a cell,thereby modulating the function of a target gene. Examples of chemokinesinclude, but are not limited to, interleukin-2 (IL-2), interleukin-10(IL-10), interleukin-17 (IL-17), tumor narcosis factor-α(TNF-α), andtumor narcosis factor-β(TNF-β). The intron RNA may contain, e.g., anantisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, or ribozyme RNA. Thecomposition can be administering into a cell according to the methodsdescribed above for modulating the function of a target gene in a cell,e.g., inducing degradation of an HIV-1 genomic sequence or preventing anHIV-1 genomic sequence from being translated into a polypeptide in anHIV-1 infected cell.

It was also found that SpRNAi-induced silencing of cellular genes Naf1β,Nb2HP and Tax1BP prevents HIV-1 type B infection. The inventiontherefore provides a composition comprising one or more agents (e.g., anantisense RNA, stRNA, miRNA, siRNA, shRNA, D-RNA, SpRNAi, ribozyme RNA,or a combination thereof) that induce RNA-mediated modulation of thefunctions of two or more target genes in a cell. The composition can beadministering into a cell according to the methods described above forcontrol of the functions of genes.

Applications of the present invention include, without limitation,therapy by suppression of cancer-related genes, vaccination againstpotential viral genes, treatment of microbe-related genes, research ofcandidate molecular pathways with systematic knockout/knockdown ofinvolved molecules, and high-throughput screening of gene functionsbased on microarray analysis. The present invention can also be used asa tool for studying gene function under physiological and therapeuticalconditions, providing compositions and methods for altering thecharacteristics of eukaryotic cells such as cancerous, virus-infected,microbe-infected, physiologically diseased, genetically mutated, andpathogenic cells.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent.

Examples 1. Cell Culture and Treatment

Rat neuronal stem cell clones AP31 and PZ5a were primary-cultured andmaintained as described by Palmer et al. (1999) J. Neuroscience 19:8487-8497. The cells were grown on polyornathine/laminin-coated dishesin DMEM/F-12 (1:1; high glucose) medium containing 1 mM·L-glutaminesupplemented with 1×N2 supplements (Gibco/BRL, Gaithersburg, Md.) and 20ng/ml FGF-2 (Invitrogen, Carlsbad, Calif.) without serum at 37° C. under5% CO₂. For long-term primary cultures, 75% of the medium was replacedwith new growth medium every 48 h. Cultures were passaged at ˜80%confluency by exposing the cells to trypsin-EDTA solution (IrvineScientific) for 1 min and rinsing them once with DMEM/F-12. Detachedcells were replated at 1:10 dilution in fresh growth medium supplementedwith 30% (v/v) conditioned medium which had been exposed to cells for 24h before passaging. Human prostatic cancer LNCaP cells were obtainedfrom American Type Culture Collection (ATCC, Rockville, Md.) and grownin RPMI 1640 medium supplemented with 10% fetal bovine serum with 100μg/ml gentamycin at 37° C. under 10% CO₂. The LNCaP culture was passagedat ˜80% confluency by exposing cells to trypsin-EDTA solution for 1 minand rinsing them once with RPMI, and detached cells were replated at1:10 dilution in fresh growth medium. After a 48-hour incubation period,RNA was isolated from the cells using RNeasy spin columns (Qiagen,Valencia, Calif.), fractionated on a 1% formaldehyde-agarose gel, andtransferred onto nylon membranes. The genomic DNA was also isolatedusing apoptotic DNA ladder kit (Roche Biochemicals, Indianapolis, Ind.)and assessed by 2% agarose gel electrophoresis, while cell growth andmorphology were examined using microscopy and cell counting.

2. Construction of SpRNAi-Containing Genes

Synthetic nucleic acid sequences used for generation of three differentSpRNAi introns containing either sense, antisense or hairpin eGFP insertwere as follows: N1-sense, 5′-pGTAAGAGGAT CCGATCGCAG GAGCGCACCATCTTCTTCAA GA-3′ (SEQ ID NO:7); N1-antisense, 5′-pCGCGTCTTGA AGAAGATGGTGCGCTCCTGC GATCGGATCC TCTTAC-3′ (SEQ ID NO:8); N2-sense, 5′-pGTAAGAGGATCCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3′ (SEQ ID NO:9); N2-antisense,5′-pCGCGTCAGGA GCGCACCATC TTCTTCAAGC GATCGGATCC TCTTAC-3′ (SEQ IDNO:10); N3-sense, 5′-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAAGTTAACTTGA AGAAGATGGT GCGCTCCTGA-3′ (SEQ ID NO:11); N3-antisense,5′-pCGCGTCAGGA GCGCACCATC TTCTTCAAGT TAACTTGAAG AAGATGGTGC GCTCCTGCGATCGGATCCTC TTAC-3′ (SEQ ID NO:12); N4-sense, 5′-pCGCGTTACTA ACTGGTACCTCTTCTTTTTT TTTTTGATAT CCTGCAG-3′ (SEQ ID NO:13); N4-antisense,5′-pGTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3′ (SEQ ID NO:14).Additionally, two exon fragments were generated by DraII restrictionenzyme cleavage of red fluorescent rGFP gene at nucleotide (nt) 208, andthe 5′ fragment was further blunt-ended using T4 DNA polymerase. TherGFP refers to a new red-fluorescin chromoprotein generated by insertionof an additional aspartate at amino acid (aa) 69 of HcRed1 chromoproteinfrom Heteractis crispa. (Gurskaya et al. (2001) FEBS Letters 507:16-20), developing less aggregate and almost twice intense far-redfluorescent emission at ˜570-nm wavelength. This mutated rGFP genesequence was cloned into pHcRed1-N1/1 plasmid vector (BD Biosciences)and propagated in E. coli DH5α in LB medium containing 50 μg/mlkanamycin (Sigma). The pHcRed1-N1/1 plasmid was cleaved with XhoI andXbaI restriction enzymes. A 769-bp rGFP fragment and a 3,934-bp emptyplasmid were purified separately from a 2% agarose gel afterelectrophoresis.

Hybridization of N1-sense to N1-antisense, N2-sense to N2-antisense,N3-sense to N3-antisense, and N4-sense to N4-antisense was separatelyperformed by heating each mixture of complementary sense and antisense(1:1) sequences to 94° C. for 2 min and then 70° C. for 10 min in 1×PCRbuffer (e.g., 50 mM Tris-HCl, pH 9.2 at 25° C., 16 mM (NH₄)₂SO₄, 1.75 mMMgCl₂). Subsequently, ligation of the N1, N2 or N3 hybrid to the N4hybrid was performed by gradually cooling a mixture of N1-N4, N2-N4 orN3-N4 (1:1) hybrids from 50° C. to 10° C. over a period of 1 h, and thenT₄ ligase and buffer (Roche) were mixed with the mixture for 12 h at 12°C. so as to obtain introns for linking to exons with proper ends. Afterthe rGFP exon fragments were added into the reaction (1:1:1), T4 ligaseand buffer were adjusted accordingly for continued ligation for another12 h at 12° C. For cloning the right sized recombinant rGFP gene, 10 ngof the ligated nucleotide sequences were amplified by PCR withrGFP-specific primers 5′-dCTCGAGCATG GTGAGCGGCC TGCTGAA-3′ (SEQ IDNO:15) and 5′-dTCTAGAAGTT GGCCTTCTCG GGCAGGT-3′ (SEQ ID NO:16) at 94°C., 1 min; 52° C., 1 min; and then 68° C., 2 min for 30 cycles. The PCRproducts were fractionated on a 2% agarose gel, and a ˜900-bp nucleotidesequence was extracted and purified using a gel extraction kit (Qiagen).The composition of this ˜900 bp SpRNAi-eGFP-containing rGFP gene wasconfirmed by sequencing.

3. Cloning of SpRNAi-Containing Genes into Various Vectors

Since the recombinant gene possesses an XhoI and an XbaI restrictionsite at its 5′- and 3′-end, respectively, it can be easily cloned into avector with ends complementary to the XhoI and XbaI sites. The vectorcan be an expression vector, e.g., a plasmid, cosmid, phagmid, yeastartificial chromosome, or viral vector. Moreover, since the insertwithin the intron is flanked by a PvuI and an MluI restriction site atits 5′- and 3′-end, respectively, the insert can be removed and replacedwith another insert with ends complementary to the PvuI and MluI sites.The insert sequence can be homologous or complementary to a genefragment such as a fluorescent protein gene, luciferase gene, lac-Zgene, plant gene, viral genome, bacterial gene, animal gene, and humanoncogene. The homology and/or complementarity ranges from about 30˜400%,more preferably 35˜49% for a hairpin-shRNA insert and 90˜100% for bothsense-siRNA and antisense-siRNA inserts.

For cloning into plasmids, the SpRNAi-recombinant rGFP gene and thelinearized 3,934-bp empty pHcRed1-N1/1 plasmid were mixed at 1:16 (w/w)ratio. The mixture was cooled from 65° C. to 15° C. over a period of 50min, and then T₄ ligase and buffer were added into the mixture forligation at 12° C. for 12 h. A so formed SpRNAi-recombinantrGFP-expressing plasmid vector was propagated in E. coli DH5α in LBmedium containing 50 μg/ml kanamycin. A positive clone was confirmed byPCR with rGFP-specific primers SEQ ID NO:15 and SEQ ID NO:16 at 94° C.,1 min and then 68° C., 2 min for 30 cycles and subsequent sequencing.For cloning into viral vectors, the same ligation procedure wasperformed except that an XhoI/XbaI-linearized pLNCX2 retroviral vector(BD Biosciences) was used. The eGFP insert within the SpRNAi intron wasremoved and replaced with various integrin β1-specific insert sequenceswith ends complementary to the PvuI and MluI sites.

Synthetic nucleic acid sequences used for generation of various SpRNAiintrons containing either sense, antisense or hairpin integrin β1 insertwere as follows P1-sense, 5′-pCGCAAGCAGG GCCAAATTGT GGGTA-3′ (SEQ IDNO:17); P1-antisense, 5′-pTAGCACCCAC AATTTGGCCC TGCTTGTGCG C-3′ (SEQ IDNO:18); P2-sense, 5′-pCGACCCACAA TTTGGCCCTG CTTGA-3′ (SEQ ID NO:19);P2-antisense, 5′-pTAGCCAAGCA GGGCCAAATT GTGGGTTGCG C-3′ (SEQ ID NO:20);P3-sense, 5′-pCGCAAGCAGG GCCAAATTGT GGGTTTAAAC CCACAATTTG GCCCTGCTTGA-3′ (SEQ ID NO:21); P3-antisense, 5′-pTAGCACCCAC AATTTGGCCC TGCTTGAATTCAAGCAGGGC CAAATTGTGG GTTGCGC (SEQ ID NO:22). These inserts weredesigned using Gene Runner software v3.0 (Hastings, Calif.) and formedby hybridization of P1-sense to P1-antisense, P2-sense to P2-antisenseand P3-sense to P3-antisense for targeting nt 244˜265 of the integrin β1gene (GenBank Access No. NM 002211.2). The SpRNAi-containingrGFP-expressing retroviral vector was propagated in E. coli DH5α in LBmedium containing 100 μg/m ampcillin (Sigma). A packaging cell line PT67(BD Biosciences) was also used for producing infectious,replication-incompetent viruses. Transfected PT67 cells were grown inDMEM medium supplemented with 10% fetal bovine serum with 4 mML-glutamine, 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate and 50mg/ml neomycin (Sigma) at 37° C. under 5% CO₂. The titer of the virusproduced by PT67 cells was determined to be at least 10⁶ cfu/ml beforetransfection.

4. Low Stringency Northern Blot Analysis

RNA (20 μg total RNA or 2 μg poly[A⁺] RNA) was fractionated on 1%formaldehyde-agarose gels and transferred onto nylon membranes(Schleicher & Schuell, Keene, N. H.). A synthetic 75-bp probe(5′-dCCTGGCCCCC TGCTGCGAGT ACGGCAGCAG GACGTAAGAG GATCCGATCG CAGGAGCGCACCATCTTCTT CAAGT-3′ (SEQ ID NO:23)) targeting the junction regionbetween rGFP and the hairpin eGFP-insert was labeled with the Prime-ItII kit (Stratagene, La Jolla, Calif.) by random primer extension in thepresence of [³²P]-dATP (>3000 Ci/mM, Amersham International, ArlingtonHeights, Ill.), and purified using 30 bp-cutoff Micro Bio-Spinchromatography columns (Bio-Rad, Hercules, Calif.). Hybridization wascarried out in a mixture of 50% freshly deionized formamide (pH 7.0),5×Denhardt's solution, 0.5% SDS, 4×SSPE and 250 mg/mL denatured salmonsperm DNAs (18 h, 42° C.). Membranes were sequentially washed twice in2×SSC, 0.1% SDS (15 min, 25° C.), and once in 0.2×SSC, 0.1% SDS (15 min,25° C.) before autoradiography.

5. Suppression of Specific Gene Expression

For interference with eGFP expression, rat neuronal stem cells weretransfected with SpRNAi-recombinant rGFP plasmids encoding either asense, antisense or hairpin eGFP insert using Fugene reagent (Roche).Plasmids containing insert-free rGFP gene and SpRNAi-recombinant rGFPgene with an insert against HIV-gag p24 were used as negative controls.Cell morphology and fluorescent images were photographed at 0-, 24- and48-hour time points after transfection. At the 48-h incubation timepoint, the rGFP-positive cells were sorted by flow cytometry andcollected for Western blot analysis. For interference with integrin β1expression, LNCaP cells were transfected with pLNCX2 retroviral vectorscontaining various SpRNAi-recombinant rGFP genes against nt 244˜265 ofintegrin β1 using the Fugene reagent. The transfection rate of pLNCX2retroviral vector into LNCaP cells was determined to be about 20%, whilethe pLNCX2 virus was less infectious to LNCaP cells. The same analyseswere performed as aforementioned.

6. SDS-PAGE and Western Blot Analysis

For immunoblotting, cells were rinsed with ice-cold PBS after the growthmedium was removed, and then treated with the CelLytic-Mlysis/extraction reagent (Sigma Chemical, St. Louis, Mo.) supplementedwith protease inhibitors, Leupeptin, TLCK, TAME and PMSF followingmanufacturer's recommendations. The cells were incubated at roomtemperature on a shaker for 15 min, scraped into microtubes, andcentrifuged for 5 min at 12,000×g to pellet the cell debris.Protein-containing cell lysate was collected and stored at −70° C. untiluse. Protein concentrations were determined as described (Bradford(1976) Anal. Biochem. 72: 248-254) using SOFTmax software package on anE-max microplate reader (Molecular Devices, Sunnyvale, Calif.). 30 μg ofcell lysate was added into SDS-PAGE sample buffer either with (reduced)or without (unreduced) 50 mM DTT, and boiled for 3 min before loadingonto 8% polyacrylamide gels, while the reference lane was loaded with2˜3 μl molecular weight markers (BioRad). SDS-polyacrylamide gelelectrophoresis was performed according to the standard protocols(Sambrook and Russell, Molecular Cloning, 3rd Ed., (2001) Cold SpringHarbor Laboratory Press: New York). Protein fractions wereelectroblotted onto a nitrocellulose membrane, blocked with Odysseyblocking reagent (Li-Cor Biosciences, Lincoln, NB) for 1˜2 h at roomtemperature. GFP expression was assessed using primary antibodiesdirected against eGFP (1:5,000; JL-8, BD Biosciences, Palo Alto, Calif.)or rGFP (1:10,000; BD Biosciences) overnight at 4° C. The blot was thenrinsed 3 times with TBS-T and exposed to a secondary antibody, goatanti-mouse IgG conjugate with Alexa Fluor 680 reactive dye (1:2,000;Molecular Probes), for 1 h at room temperature. After three more TBS-Trinses, scanning and image analysis were performed using Li-Cor OdysseyInfrared Imager and Odyssey Software v.10 (Li-Cor). For integrin β1analysis, the same procedure was performed except that primaryantibodies directed against integrin β1 (1:2,000; LM534, Chemicon,Temecula, Calif.) were used.

7. In Vitro Generation of Deoxyribonucleotidylated RNA Probes

The RNA-polymerase cycling reaction (RNA-PCR) procedure can be modifiedto synthesize mRNA-aDNA and/or mDNA-aRNA hybrids (Lin et al. (1999)Nucleic Acids Res. 27, 4585-4589) from an SpRNAi-recombinant gene,expression-competent vector template or transcriptome source. As anexample of using an SpRNAi-recombinant gene source, an SpRNAi-sense HIVrecombinant gene containing a sequence homologues to HIV-1 genome from+2113 to +2453 bases was generated following a procedure similar toSection 2 above. The RNA product (10˜50 μg) of the SpRNAi-sense HIVrecombinant gene were transcribed in about 10⁶ transfected cells,isolated using RNeasy columns (Qiagen), and then hybridized to itspre-synthesized complementary DNA (cDNA) by heating and then cooling themixture from 65° C. to 15° C. over a period of 50 min. Transfection wasperformed following the same procedure shown in Section 5 above.

8. Design of Artificially Recombined Genes for Splicing-Directed GeneSilencing

RNA splicing/processing-directed gene silencing was tested using anartificial recombinant gene, SpRNAi-rGFP (FIG. 1). A DNA template for asplicing-competent intron (SpRNAi) was inserted into an intron-free redfluorescin gene (rGFP), providing splicing-directed gene silencingeffects through pre-mRNA splicing and some unknown processingmechanisms. Although a model of gene silencing through pre-mRNA splicingis shown here, the same principle can be used for the design of genesilencing inserts working through other pre-RNA processing, e.g.,pre-ribosomal RNA (pre-rRNA)-processing, which is mainly mediated bytype-I RNA polymerase (Pol I) transcription machinery. Thesplicing-competent intron is flanked by a donor (DS) and an acceptor(AS) splicing site, and contains at least one gene homologue insert,branch point (BrP) and poly-pyrimidine tract (PPT) inbetween the DS-ASsites for interacting with spliceosome machinery. Using low stringencyNorthern blotting (mid-bottom of FIG. 1), 15˜45 bp intron-insertfragments were seen to be released from the SpRNAi-rGFP gene transcript(left), rather than an intron-free rGFP (middle) or a defectiveSpRNAi-rGFP (right) RNA without a functional splice donor site, whilethe spliced exons were linked to form a mature RNA for rGFP proteinsynthesis. The “?” mark in FIG. 1 indicates an unknown mechanism forprocessing of a ˜120-bp intron, resulting in small interferingintron-insert fragments. Short sense, short antisense and hairpinconstructs of some gene homologue inserts were successfully tested forinducing specific gene silencing in various cell types.

As shown in FIG. 1, DNA templates for splicing-competent introns(SpRNAi) were synthesized and inserted into an intron-free redfluorescin gene (rGFP) that was mutated from the HcRed1 chromoprotein ofHeteractis crispa. Since the inserted intron disrupted the functionalfluorescin structure of the rGFP protein, occurrence of intron splicingand rGFP-mRNA maturation was indicated by the reappearance of redfluorescent light emission at the 570-nm wavelength in a transfectedcell. Construction of SpRNAi was based on the natural structure of apre-messenger RNA intron, consisting of spliceosome-dependent nucleotidecomponents, such as donor and acceptor splicing sites in both ends forprecise cleavage, branch point domain for splicing recognition,poly-pyrimidine tract for spliceosome interaction, linkers forconnection of each major components and some artificially added multiplerestriction/cloning sites for cloning of inserts. Based on priorstudies, the donor splicing site is an oligonucleotide sequence eithercontaining or homologous to 5′-exon-AG-(splicingpoint)-GTA(A/-)GAG(G/T)-3′ (SEQ ID NO:24), e.g., 5′-AG GTAAGAGGAT-3′(SEQ ID NO:25), 5′-AG GTAAGAGT-3′ (SEQ ID NO:26), 5′-AG GTAGAGT-3′,5′-AG GTAAGT-3′ and so on. The acceptor splicing site is anoligonucleotide sequence either containing or homologous to5′-G(W/-)(T/G)(C/G)C(T/C)(G/A)CAG-(splicing point)-G/C-exon-3′ (while Wis a pyrimidine, i.e., A or T) (SEQ ID NO:27), e.g., 5′-GATATCCTGCAGG-3′ (SEQ ID NO:28), 5′-GGCTGCAG G-3′, 5′-CCACAG C-3′ and so on. Thebranch point is an “A” residue located within a sequence homologous to5′-TACT(A/T)A*(C/T)(-/C)-3′ (while the symbol “*” marks the branchsite), e.g., 5′-TACTAAC-3′, 5′-TACTTATC-3′ and so on. Thepoly-pyrimidine tract is a high T and/or C content oligonucleotidesequence homologous to 5′-(TY)m(C/-)(T)nC(C/-)-3′ or5′-(TC)nNCTAG(G/-)-3′ (while Y is a C or T). The symbols “m” and “n”indicate the numbers of repeats, preferably, m=1˜3 and n=7˜12. For allthe above splicing components, the deoxythymidine (T) in a DNA templateis replaced by uridine (U) after transcription.

To test the function of a spliced intron, various inserts were clonedinto SpRNAi through multiple restriction/cloning sites, e.g., those forAatII, AccI, AflII/III, AgeI ApaI/LI, AseI, Asp718I, BamHI, BbeI,BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI,BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II,EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI,HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI,NcoI, NdeI, NgoMI, NotI, NruI, NsiI, Pm1I, Ppu10I, PstI, PvuI/II, RsaI,SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI,XhoI and/or XmaI endonucleases. These inserts are DNA templates foraberrant RNAs, e.g., short-temporary RNA (stRNA), small-interfering RNA(siRNA), short-hairpin RNA (shRNA), long deoxyribonucleotide-containingRNA (D-RNA) and potentially ribozyme RNA in either sense or antisenseorientation. As demonstrated in the examples below, the gene silencingeffect of a hairpin-RNA-containing SpRNAi is stronger than that of asense- and antisense-RNA-containing SpRNAi, showing an average of >80%knockdown specificity to all targeted gene products. Such knockdownspecificity is mainly determined by the homologous or complementaryregion of an insert to the targeted gene transcript. For example, thetested hairpin-SpRNAi insert had about 40˜42% homology and another40˜42% complementarity to the targeted gene domain, with-in-between ofwhich an A/T-rich linker sequence filled in the rest 8˜10% space. Forthe less potent sense- and antisense-SpRNAi inserts, although thehomology or complementarity can be increased up to 100%, an average of40˜50% knockdown efficacy was detected in most of the transfectiontests. Thus, different types of SpRNAi inserts and/or the combinationthereof can be used to manipulate specific gene expression levels incells.

9. Simultaneous Expression of rGFP and Silencing of eGFP by SpRNAiTransfection

For the convenience of gene delivery and activation in cells,SpRNAi-containing genes were cloned into an expression-competent vector,e.g., plasmid, cosmid, phagmid, yeast artificial chromosome, viralvector and so on. As shown in FIGS. 1-4, the vectors can contain atleast one viral or type-II RNA polymerase (Pol II) promoter or both forthe expressing of the SpRNAi-gene in eukaryotic cells, a Kozak consensustranslation initiation site to increase translation efficiency ineukaryotic cells, SV40 polyadenylation signals downstream of theSpRNAi-gene for processing of the 3′-end gene transcript, a pUC originof replication for propagation in prokaryotic cells, at least twomultiple restriction/cloning sites for cloning of the SpRNAi-gene, anoptional SV40 origin for replication in mammalian cells expressing theSV40 T antigen and an optional SV40 early promoter for expressing anantibiotic resistance gene in replication-competent prokaryotic cells.The expression of antibiotic resistance genes was used as a selectivemarker for searching of successfully transfected or infected clones thatare resistance to the antibiotics such as penicillin G, ampcillin,neomycin, paromycin, kanamycin, streptomycin, erythromycin,spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B,gentamicin, chloramphenicol, cephalothin, tylosin and the combinationthereof. The vector was therefore stable enough to be introduced into acell(s), tissue or animal body using a highly efficient gene deliverymethod, e.g., liposomal transfection, chemical transfection, chemicaltransformation, electroporation, infection, micro-injection and gene-gunpenetration.

As shown in FIG. 5, transfection of the plasmids described in Sections 2and 3 (containing various SpRNAi-rGFP recombinant genes against theexpression of a commercially available Aequorea victoria greenfluorescent protein (eGFP)) was found to be successful in bothexpression of rGFP (red) and silencing eGFP (green). The use ofeGFP-positive rat neuronal stem cell clones provided an excellent visualaid to measure the silencing effects of various SpRNAi inserts. Ratneuronal stem cell clones AP31 and PZ5a were primary-cultured andmaintained as described in Section 1. 24-h after transfection, almostthe same amount of total cell number and eGFP-positive cell populationwere well seeded and very limited apoptotic or differentiated cellsoccurred. Silencing of eGFP emission at the 518-nm wavelength wasdetected 36˜48 hours after transfection, indicating a potential onsettiming required for the release of small interfering inserts fromSpRNAi-rGFP gene transcripts by spliceosome machinery. Since allsuccessfully transfected cells displayed red fluorescent emission atabout 570-nm wavelength, the gene silencing effect was traced bymeasuring relative light intensity of the green fluorescent emission inthe red fluorescent cells, showing a knockdown potency ofhairpin-eGFP>>sense-eGFP^(˜)antisense-eGFP>>hairpin-HIV p24 (negativecontrol) insert.

10. Western Blot Analysis of RNA Splicing/Processing-Directed eGFPSilencing

As shown in FIG. 6, knockdown levels of eGFP protein in rat neuronalstem cell clones AP31 and PZ5a by various SpRNAi inserts were measuredon an unreduced 6% SDS-polyacrylamide gel. To normalize the loadingamounts of transfected cellular proteins, rGFP protein levels (˜30 kDa,red bars) were adjusted to be comparatively equal, representing anaverage expression range of 82˜100% intensity (Y axis). The eGFP levels(27 kDa, green bars) were found to be reduced by transfection ofSpRNAi-rGFP genes containing sense-eGFP (43.6%), antisense-eGFP (49.8%)or hairpin-eGFP (19.0%) inserts, but not that of intron-free rGFP gene(blank control) or SpRNAi-rGFP gene containing hairpin-HIV p24 insert(negative control). These findings confirm the knockdown potency ofhairpin-eGFP>>sense-eGFP^(˜)antisense-eGFP>>hairpin-HIV p24 (negativecontrol), and also demonstrate that only a gene insert which is eitherhomologous or complementary (or partially homologous or complementary)to the targeted gene can elicit this gene-specific silencing effect.

11. Western Blot Analysis of RNA Splicing/Processing-Directed Integrinβ1 Silencing

As shown in FIG. 7, a similar splicing/processing-directed genesilencing phenomenon was seen in human cancerous LNCaP cells. Knockdownlevels of integrin β1 (ITGb1) protein by various SpRNAi inserts, weremeasured on a reduced 8% SDS-polyacrylamide gel. The relative amounts ofrGFP (black bars), ITGb1 (gray bars) and actin (white bars) were shownon a percentage scale (Y axis). The ITGb1 levels (29 kDa) weresignificantly reduced by transfection of SpRNAi-rGFP genes containing asense-ITGb1 (37.3%), antisense-ITGb1 (48.1%) and hairpin-ITGb1 (13.5%)inserts, but not that of an intron-free rGFP gene (blank control) orSpRNAi-rGFP gene containing a hairpin-HIV p24 insert (negative control).Co-transfection of SpRNAi-rGFP genes containing sense- andantisense-ITGb1 inserts elicited a significant gene silencing effect(22.5%) and 10˜15% cell death, while that of SpRNAi-rGFP genescontaining hairpin-ITGb1 and hairpin-p58/HHR23 inserts partially blockedthe splicing-directed gene silencing effect and resulted in an averageof 57.8% expression level. These findings indicate that theSpRNAi-induced gene silencing effects may work on a wide range of genesand cell types of interest.

12. Combinational Therapy for HIV Eradication and Vaccination

The ex vivo transfection of a viral RNA-antisense DNA hybrid constructin conjunction with interleukin 2 adjuvant therapy was found to silencean average of 99.8% human immunodeficiency virus-1 (HIV-1) subtype Bgene activity through a novel posttranscriptional gene silencingmechanism, deoxyribonucleotidylated RNA interference (D-RNAi; Lin et al.(2001) supra). This combined therapy not only delivered a strongsuppression effect on viral replication but also boosted the immunityand proliferation of non-infected CD4⁺ T lymphocytes. A normal T celloutgrowth effect was observed to achieve maximal 76.2% HIV-infected cellelimination after one-week of therapy. RNA-directed endoribonucleaseactivity was mildly increased up to 6.7% by the transfection, while nointerferon-induced cytotoxicity was detected. The cellular genescorresponding to combinational therapy have been, further investigatedby microarray analysis for AIDS prevention. Co-suppression of threemicroarray-identified target genes, Naf1β, Nb2 protein homologous toWnt-6 and Tax1 binding protein was shown to prevent an average of 80.2%HIV-1b entry and infection in a primary CD4⁺ T cell model. Thesefindings indicate an immediate therapy in both acute and chronic HIV-1infections and also a potential vaccination useful for AIDS elimination.

In order to test the effectiveness of D-RNAi to inactivate HIV-1replication, a viral RNA (vRNA)-antisense DNA (aDNA) hybrid constructwas designed to silence an early-stage gene locus containing gag/pol/proviral genes and p24 HIV-1 gene marker. The anti-gag/pol/pro transfectioninterferes with the integration of viral provirus into host chromosomeand also prevents the activation of several viral genes, while theanti-p24 transfection provides a visual indicator for observing viralactivity on an ELISA assay. The results showed that such strategy waseffective in knocking out exogenous viral gene expression ex vivo in aCD4⁺ T lymphocyte extract model. Peripheral blood mononuclear cells(PBMC) extracted from patients were purified using CD4⁺-affinityimmunomagnetic beads and grown in RPMI 1640 medium with 200 U/ml IL-2adjuvant treatment for more than two weeks. A vRNA-aDNA hybrid probecontaining partial HIV genomic sequence from +2113 to +2453 bases wasgenerated using a pre-designed SpRNAi-recombinant gene (used as acontrol as described in previous sections) homologous to gag-p24 genes.After 96-h incubation, the expression activity of HIV-1 genome wasmeasured by Northern blotting and found to be almost completely shutdown in the D-RNAi hybrid transfection sets.

The gene silencing effects of anti-HIV D-RNAi transfections in the acutephase AIDS patient T lymphocyte extracts were biostatisticallysignificant (n=3, p<0.01). Pure HIV-1 provirus was shown as a viralgenome sized about 9.7 kb on a formaldehyde-containing RNAelectrophoresis gel. Samples of CD4⁺ Th lymphocyte RNA extracts fromnormal, non-infected persons were used as negative controls, while thosefrom HIV-1 infected patients were used as positive controls. Nosignificant gene silencing effect was detected in all controls ortransfections of other constructs, including vDNA-aRNA hybrid of HIV-1b,aDNA only and vRNA-aDNA against HTLV-1 rather than HIV-1. In the acutephase (<2-week infection), treatment with 5 nM D-RNAi knocked out anaverage of 99.8% viral gene expression, whereas in the chronic phase(˜two-year infection), the same treatment knocked down only an averageof 71.4% viral gene expression. Although higher RNase activities werefound in chronic HIV-1⁺ T cells by microarray analysis, transfection ofD-RNAi in higher concentration (more than 25 nM) can overcome this drugresistance. Unlike dsRNA, transfection of highly concentrated vRNA-aDNAhybrids did not cause significant interferon-induced cytotoxic effects,because the house-keeping gene, β-actin, was expressed normally in allsets of cells. Since the Northern blot method is able to detect HIV-1gene transcript at the nanogram level, the above strong viral genesilencing effect suggests a very promising pharmaceutical andtherapeutical potential for combinational administration of D-RNAi andIL-2 as antiviral therapy and/or vaccination.

Northern blot analysis of SpRNAi-induced gene silencing effects onNaf1β, Nb2HP and Tax1BP was shown to prevent HIV-1 type B infection(FIG. 8). The tested gene targets were selected through RNA-PCRmicroarray analysis of differential expression genes from the acute(one˜two weeks) and chronic (about two years) infected patients' primaryT cells with or without 25 nM anti-HIV D-RNAi treatment (Lin et al.(2001) supra). The SpRNAi product concentrations in all treatments werenormalized to 30 nM. FIG. 8B is a bar chart showing HIV-gag p24 ELISAresults (white) in correlation with the treatment results demonstratedin FIG. 8A.

In view of CD4 function in IL-2 stimulation and T-cell growth andactivation, CD4 may not be an ideal target for HIV prevention. However,the search for HIV-dependent cellular genes in vivo was hindered by thefact that infectivity of viruses and infection rate among differentpatients are usually different and lead to inconsistent results.Short-term ex vivo culture conditions can normalize infectivity andinfection rate of HIV transmission in a more uniformed CD4⁺ T cellpopulation. Microarray analysis based on such ex vivo conditions wouldbe reliable for critical biomedical and genetic research of HIV-1infection. Microarray studies identified differential gene profilesbetween HIV⁻ and HIV⁺ T cells in the acute and chronic infection phasesand provided many potential anti-HIV cellular gene targets for AIDStherapy and prevention. To functionally evaluate the usefulness oftargeting cellular genes for HIV vaccination, three highlydifferentially expressed genes, Naf1β, Nb2 homologous protein to Wnt-6(Nb2HP) and Tax1 binding protein (Tax1BP) were tested for inhibitingHIV-1 infection. Since each of these genes contributes to only a part ofAIDS complications, knockdown of a single target gene failed to suppressHIV-1 infection, while combination of all three SpRNAi probes at thesame total concentration showed a significant (80±10%) reduction inHIV-1b infection (FIG. 8A, n=3, p<0.01). The ELISA results of HIVgag-p24 protein (FIG. 8B) also correlated with the Northern blot data,showing an average of 77±5% reduction of gag-p24 expression. Thesefindings indicate the feasibility of a novel strategy for retroviralvaccination using PTGS mechanisms against cellular target genes.

Two major phenomenal differences between PTGS/RNAi and SpRNAi mechanismswere found. First, the onset of SpRNAi effects takes a period of timemore than 36-48 hours, which is longer than the timing needed for theonset of PTGS/RNAi (12-24 hours). Second, although the role ofPTGS/RNAi-associated Dicer enzymes is unclear for the SpRNAi-directedgene silencing mechanism, several repair complementing antigens werefound to be involved. Homologous recombination machinery involvingnucleotide excision repair-related gene p58/HHR23 was found to play apotential role of Dicer in SpRNAi induction. The p58/HHR23 species thatcodes for XP-C repair-complementing proteins is a human homologue ofyeast RAD23 derivatives, sharing an ubiquitin-like N-terminus. Based onits molecular similarity shared with RNA repairing-directedtranscriptional regulation, the repair-complementing machinery indicatesa novel mechanism of posttranscriptional gene silencing in addition toRNA interference.

Homologous recombination between intracellular mRNAs and the RNAcomponents of a D-RNAi agent construct probably accounted for itsspecific gene silencing effect (Lin et al. (2001) Current Cancer DrugTargets 1: 241-247). [P³²]-labeled DNA component of a D-RNAi agentconstruct was found to be intact in a hybrid duplex during the effectiveperiod of a D-RNAi phenomenon, while the labeled RNA part was replacedby a cold homologue and degraded into small RNA oligoribonucleotideswithin a 3-day incubation period (FIG. 10A). It is possibly that theD-RNAi agent can facilitate the degradation of non-recombined parts ofits mRNA homologue as shown in FIG. 10B. Alternatively, the newlyrecombined mRNA part of the D-RNAi agent may be further processed byintracellular Pol II and some unknown RNA excision machineries togenerate miRNA-like molecules for long-term gene silencing. This issupported by the fact that both D-RNAi-derived small RNAs and Pol II RNAsplicing-processed intron fragments have an average length of 15-45nucleotides, which is comparable to the general sizes of Dicer-processedmiRNA intermediates. Additionally, both kinds of small RNAs isolated byguanidinium-chloride ultracentrifugation can elicit strong, butshort-term gene silencing effects to genes homologous to the small RNAsin cells, indicating the possible miRNA-related interfering property ofthese small RNAs. Since the small miRNA-like RNAs are constitutionallyderived from the large templates of mRNA-cDNA or precursor mRNA-genomicDNA hybrids, the long-term effect of D-RNAi phenomena may be maintainedby accumulation of sufficient small miRNA-like RNAs rather than thestability of small RNAs. This also explains the delayed initiation phaseobserved in the D-RNAi-induced gene silencing and intronsplicing-mediated PTGS phenomena (Lin et al. (2001) Biochem. Biophys.Res. Commun. 281: 639-644; and Lin et al. (2003) Biochem. Biophys. Res.Commun., in press).

Previous studies (Zhang et al. (1994) Nature 372: 809-812; and Ghosh andGarcia-Blanco (2000) RNA 6: 1325-1334) have demonstrated that a coupledinteraction between nascent Pol II pre-mRNA transcription and intronexcision occurs within certain nuclear region proximal to genomic DNA(i.e., perichromatin fibrils), indicating a reasonable potential forD-RNAi-associated miRNA generation in cells. The spliced introns are notcompletely digested into monoribonucleotides for transcriptionalrecycling, as approximately 10˜30% of the introns are found in thecytoplasm with a moderate half-life (Nott et al. (2003) RNA 9: 607-617).In an effort to examine such a process, an artificial intron mimickingthe natural structure of a pre-mRNA intron was constructed forevaluating splicing-directed small RNA generation (Lin et al. (2003)Biochem. Biophys. Res. Commun., in press). The splicing-competentartificial intron, SpRNAi, is flanked by a splice donor (DS) andacceptor (AS) site, and contains a branch-point domain (BrP), apoly-pyrimidine tract (PPT) and at least one intronic insert located inthe 5′-proximal domain of the artificial intron. To ensure the accuracyof pre-mRNA splicing, the SpRNAi also contains a translation stop codonin its 3′-proximal region, which if present in a cytoplasmic mRNA, wouldsignal the diversion of the defective pre-mRNA from a non-sense mRNAdegradation (NMD) pathway. As shown by results from low stringencyNorthern blotting, the intracellular processing of a spliced intron intosmall RNA fragments was found to be highly efficient. The release ofsmall 15˜45 nt RNA fragments was found to be only from theintron-containing gene transcripts, but not from an intronless mRNA or asplice-donor-defective pre-mRNA (a positive example of NMD). The smallmiRNA-like RNAs are able to trigger translation repression or sometimesRNA degradation depending on the degree of complementarity and homologywith their targets. According to the variety and complexity of naturalmiRNA structures, there is no artificial means to produce intracellularmiRNA-like molecules before the finding of this intron splicing-mediatedgene silencing phenomenon. The process of such miRNA-like smallinterfering RNA generation is therefore different from that for thedsRNA-induced RNAi; however, the possible involvement of RNAi mechanismscannot be ruled out in that some small RNAs might form siRNAs bycomplementary hybridization within a localized compartment.

13. In Vivo Gene Silencing Using D-RNAi Agents

D-RNAi can be used as an effective strategy to silence specific targetgene in vivo. β-catenin gene was selected as an example because itsproduct plays a critical role in the biological development andontogenesis. β-catenin is known to be involved in the growth control ofskin and liver tissues in chicken embryos. As shown in FIG. 11,experimental results demonstrated that D-RNAi (mRNA-cDNA hybrid) agentswere capable of inhibiting β-catenin gene expression in the liver andskin of developing chicken embryos. The anti-β-catenin D-RNAi moleculeswere generated against the central region (aa 306-644) of the β-catenincoding sequence (GenBank Access No. X87838) by RNA-PCR. Fertilized eggswere obtained from SPAFAS farm (Preston, Conn.) and incubated in ahumidified incubator. Using embryonic day 3 chicken embryos, a dose of25 nM of the D-RNAi agent or reversal control hybrids of senseDNA-antisense RNA (sDNA-aRNA) was injected into the ventral body cavity,which is close to where the liver primordia would form (FIG. 11A). ThemRNA-cDNA or the control sDNA-aRNA hybrids were mixed with DOTAP®liposomal transfection reagent (Roche) at a ratio of 3:2. A 10% (v/v)fast green solution was added before injection as a dye indicator. Themixtures were injected into the ventral side near the liver primordiaand below the heart using heat pulled capillary needles. Afterinjection, the eggs were sealed with scotch tape and put back into thehumidified incubator at 39-40° C. until day 12 when the embryos wereremoved, examined and photographed under a dissection microscope. Whilethere were malformations, the embryos survived and there was no visibleovert toxicity or overall perturbation of embryo development. The liverwas the closest organ to the injection site and was most dramaticallyaffected in its phenotypes. Other regions, particularly the skin, werealso affected by the diffused D-RNAi agent. As shown in FIG. 11B,Northern blot hybridizations using RNA from dissected livers showed thatβ-catenin in the control livers remained expressed (lanes 4-6), whereasthe level of β-catenin mRNA was decreased dramatically (lanes 1-3) aftertreatment with the D-RNAi agent directed against β-catenin. Controlsused included liposome alone (lane 4) and of control sDNA-aRNA hybridsin similar concentrations (lanes 5 and 6).

After ten days of injection with the anti-β-catenin D-RNAi (mRNA-cDNAhybrid) agent, the embryonic chicken livers showed an enlarged andengorged first lobe, but the size of the second and third lobes of thelivers were dramatically decreased (FIG. 11C). Histological sections ofnormal livers showed hepatic cords and sinusoidal space with few bloodcells. In anti-β-catenin D-RNAi-treated embryos, the generalarchitecture of the hepatic cells in lobes 2 and 3 remained unchanged;however, there were islands of abnormal regions in lobe 1. Theendothelium development appeared to be defective and blood leakedoutside of the blood vessels. Abnormal types of hematopoietic cells werealso observed between hepatocytes, particularly dominated by apopulation of small cells with round nuclei and scanty cytoplasm. Inseverely affected regions, hepatocytes were disrupted (FIG. 11C, smallwindows). These results showed that the anti-β-catenin D-RNAi agent wasvery effective in knocking out the targeted gene expression at a verylow dose of 25 nM and was effective over a long period of time (>10days). Furthermore, the anti-β-catenin D-RNAi gene silencing effectappeared to be very specific, as non-targeted organs appeared to benormal, indicating that the D-RNAi hybrid compositions had no overttoxicity. The gene silencing in chicken and mice by the D-RNAi agent(FIGS. 11 and 12) presents a great potential of localized transgene-likeapproach in creating animal models for human diseases.

To test in an adult animal model (FIG. 12), patched albino (white) skinsof melanin-knockout mice (Rosa-26 strain) were created by a successionof intra-cutaneous (i.c.) transduction of about 50 nM anti-tyrosinase(tyr) mRNA-cDNA hybrids for 4 days (a total of 200 nM). Tyr, a type-Imembrane protein and copper-containing enzyme, catalyzes the criticaland rate-limiting step of tyrosine hydroxylation in the biosynthesis ofmelanin (black pigment) in skins and hairs. After 14-day incubation, theexpression of melanin was blocked due to the loss of its intermediatesresulted from the tyr silencing effect. In contrast, the blank controland dsRNA-transfected mice showed normal skin color (black), indicatingthat the loss of melanin is specifically caused by the mRNA-cDNAtransfection. Moreover, Northern blot analysis using RNA-PCR-derivedmRNAs from hair follicles showed a 76.1±5.3% reduction in tyr expression2 days after the transfection of the D-RNAi agent, which was consistentwith the immunohistochemistry results from the same skin area, whereasmild, non-specific degradation of common gene transcripts was detectedin the dsRNA-transfected skins, shown by the smearing patterns of boththe house-keeping control GAPDH and tyr mRNAs in Northern blots (4^(th)column, left-bottom insert windows). These results show that theutilization of D-RNAi agents provides a powerful new strategy for invivo gene therapy, potentially to melanoma. At the same dosage (200 nMin total), the D-RNAi transfections did not cause detectablecytotoxicity, while the dsRNA transfections induced noted non-specificmRNA degradation. This even underscores the fact that the mRNA-cDNAhybrids are effective even under in vivo systems without theside-effects of dsRNA. The results also indicate that this genesilencing effect is stable and efficient in knocking out target geneexpression over a relatively long period of time since the hair regrowthrequires at least a ten-day recovery. Further, it was observed thatnon-targeted skin hairs appeared to be normal, indicating that thecompositions used herein possess high specificity and no overt toxicity.Thus, the D-RNAi-based gene manipulation offers the advantages of low invivo dosage, stability, long-term effectiveness, and lack of overttoxicity.

OTHER EMBODIMENTS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated RNA comprising an intron RNA that is released in a cell,thereby modulating the function of a target gene, wherein the isolatedRNA does not contain a combination of a splice donor site that includes5′-GU(A/G)AGU-3′ and a splice acceptor site that includes5′-CU(A/G)A(C/U)NG-3′. 2-57. (canceled)